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Molecular Basis Of Disease > Structural Chromosomal Abnormalities > Flashcards

Structural Chromosomal Abnormalities Flashcards

1
Q

State the 6 major types of structural abnormalities that can arise?

A
  • Translocations: Reciprocal, Robertsonian
  • Inversion
  • Deletion
  • Duplication
  • Rings
  • Isochromosomes
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2
Q

What is translocation?

A
  • Exchange of two segments between non-homologous chromosomes (not paired)
  • Occurs via inappropriate non-homologus end joining (NHEJ)
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3
Q

How does NHEJ translocation arise?

A
  • DNA repair mechanism
  • Common for double strands break to occur in chromosomes causing chunks to split off
  • NHEJ will rejoin the chunk back to the original chromosome
  • Inappropriate NHEJ will join the segment to another chromosome and vice versa forming derivative chromosomes
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4
Q

What is the common result of inappropriate non-homologous end joining?

A
  • Carriers of balanced translocation (no. clinical effect)
  • Forms 2 derivative chromosomes + copies of both normal chromosome (due to HC)
  • No net gain or loss of genetic materal (any chromosome and size fragment), just material is in wrong place
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5
Q

Give an example of a carrier of balanced translocation which have adverse effects?

A
  • One that forms the philadelphia chromosome (Cr 9- ABL +Cr 22-BCR)
  • Learn below VD
  • Can result in fusion gene leading to leukaemia
  • Diagnosed via G-banding
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6
Q

How are unbalanced individuals produced?

A

When they have < or > of a particular region of a chromosome

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7
Q

What is reciprocal translocation and state the common problem?

A
  • Reciprocal translocation is a form of gene rearrangement where portions of two chromosomes are simply exchanged with no net loss of genetic information
  • Common problem arise when the derivative chromosomes align with their homologue as some may contain 2 copies
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8
Q

State the consequence of the reciprocal translocations in this picture? VD important

A
  • Individual has a mixture of trisomic + monosomic with respect to chromosome
  • 3 copies of purple so trisomic
  • Tetravalent formed (4 chromosomes)
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9
Q

State the common results of unbalanced reciprocal translocation (3)?

A
  • Many lead to miscarriage (hence why a woman with a high number of unexplained miscarriages should be screened for a balanced translocation)
  • Learning difficulties, physical disabilities
  • Tend to be specific to each individual so exact risks and clinical features vary
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10
Q

What is robertsonian translocation?

A
  • Two acrocentric chromosomes join via q arms (long) at centromere with the loss of p/satellites arms (short)
  • Balanced carrier has 45 chromosomes - with one tetravalent formed
  • If 46 chromosomes present including Robertsonian then must be unbalanced - as you’ll lose a chromosome where the 2 A chr. combine together.
  • p arms encode rRNA (multiple copies so not deleterious to lose some)
  • Only involved in acrocentric chromosome
  • P arms of both chromosomes are removed, Q arm of both chromosomes combine to form robertsonian translocation
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11
Q

State which chromosomes are common for robertsonian translocations
to occur and what can it potentially lead to?

A
  • Robertsonian translocations 13;14 and 14;21 relatively common.
  • 21;21 translocation leads to 100% risk of Down syndrome in fetus
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12
Q

Give the possible gemetes and outcomes that can be formed from this

A

VD

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13
Q

What are the 5 main outcomes of translocations?

A
  • Very difficult to predict
  • Only have approximate probability of producing possible gametes
  • Some unbalanced outcomes may lead to spontaneous abortion of conceptus so early that not seen as problem
  • Some unbalanced outcomes may lead to miscarriage later on and present clinically
  • Some may result in live-born baby with various problems
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14
Q

State the two types of deletion and the effects?

A
  • Deletion may be terminal (loss of telomere) or interstitial (middle segment)
  • Causes a region of monosomy - copies of particular region in only one chromosome
  • Haploinsufficiency of some genes - The situation that occurs when one copy of a gene is inactivated or deleted and the remaining functional copy of the gene is not adequate to produce the needed gene product to preserve normal function
  • Contiguous gene syndrome (multiple, unrelated clinical features): Due to deletion at different chromosomes potentially
  • Phenotype is specific for size and place on deletion
  • Gross deletions seen on metaphase spread on G-banded karyotype
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15
Q

State how microdeletions can be seen?

A
  • Many patients had no abnormality visible on metaphase spread
  • High resolution banding, FISH and now CGH showed ‘micro’ deletions
  • Only a few genes may be lost or gained
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16
Q

State how gross deletions can be seen?

A

Metaphase spread on G-banded karyotype

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17
Q

State 4 examples of diseases that can arise from microdeletions including the chromosomes?

A
  • Velocardiofacial (DiGeorge), 22q11
  • Wolf-Hirschhorn, 4p16
  • Williams, 7q11
  • Smith-Magenis, 17p11
18
Q

State 3 examples of diseases that can arise from interstital deletion?

A
  • Prader-Willi
  • DiGeorge Syndrome
  • Cri du chat
19
Q

What mechanism results in (micro) deletions and (micro) duplications?

A
  • Unequal crossing over/ non-allelic homologous recombination
  • Unequal alignment during crossing over
  • Potential deletion or duplication of genes occuring
20
Q

State prenatal (3) and postnatal (2) sources for detecting structural abnormalities?

A
  • Prenatal
  • Amniocentesis - sample of amniotic fluid for fetal DNA
  • Chorionic villus sampling: Piece of placenta taken for access to fetal DNA
  • Cell-free fetal DNA: Not invasive, above two are
  • Postnatal: Blood, Saliva
21
Q

Describe G-banding

A
  • G = Giemsa
  • Main Purpose: How does karyotype of patient differ from expected?
  • Looks for aneuploidies, translocations & very large deletions
22
Q

Describe the point of the G banding technique

A
  • Key points of technique:
  • Uses a chemical stain (Giemsa)
  • Uses metaphase chromosomes - DNA is densely packed
  • Takes several days at least
23
Q

Why are bands produced in G-banding?

A
  • Chromatin - 2 different sorts: euchromatin & heterochromatin
  • Euchromatin = GC-rich; loosely packed; genes active = Light
  • Heterochromatin = AT-rich; tightly packed; genes inactive = Dark
  • Stain differently
24
Q

Describe the process of staining in G banding

A
  • 5 mL venous blood -> Add phytomagglutinin and culture medium -> culture at 37° for 3 days -> Add Colchicine and hypotonic saline -> Cells fixed -> Spread cells into slide by dropping -> Digest with trypsin and stain with giemsa -> Analyse metaphase spread
  • Quite a slow process
25
Q

Describe FISH

A
  • Fluorescent in situ hybridisation
  • Main purpose: How does karyotype of patient differ from expected? Looks for aneuploidies, translocations & large deletions
  • Cultured cells, metaphase spread (chromosomes) used
26
Q

Describe the process of the FISH technique?

A
  1. Fluorescent probe - for specific parts of the genome
  2. Denature probe and target DNA
  3. Mix probe and target DNA
  4. Probe binds to target - visualise using fluorescent
    - Takes several days
27
Q

What is hybridisation?

A

Hybridisation = single stranded nucleic acid binds to a new single stranded nucleic acid (DNA/DNA or DNA/RNA)

28
Q

What is a probe?

A
  • A single stranded DNA (or RNA) molecule - complementary to target DNA
  • Typically 20 - 1000 bases in length
  • Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
  • In some techniques thousands or millions of probes are used simultaneously
29
Q

State two diseases that FISH can be used for?

A
  • Cri-du-chat (5p-syndrome)
  • 22q deletion syndrome
30
Q

Describe the purpose of the array CHG technique

A
  • Array comparative genomic hybridisation
  • Main purpose: How many copies of a particular genomic region does the patient have +
  • Detection of sub-microscopic chromosomal abnormalities:
    microdeletions and microduplications
31
Q

Describe the process of the Array CGH technique

A
  • Uses fluorescent probes to differentiate between patient and control
  • Patient DNA is labelled green and control DNA is labelled red.
  • Uses extracted DNA
32
Q

Describe the purpose of the Qf-PCR technique

A
  • Quantitive fluorescence polymerase chain reaction
  • Main purpose: How many copies of a chromosome does the patient have?
  • Looks for aneuploidies (trisomies 13,18 and 21)
33
Q

Describe the process of the QF-PCR technique

A
  • Uses fluorescent probes for SPECIFIC microsatellite markers on SPECIFIC chromosomes
  • Uses extracted DNA
  • Quick (~48hrs)
  • Need to know what MS you’re looking for
34
Q

What are microsatellites?

A
  • Short repeated sequences
  • Number of repeats varies between individuals
  • Total length of microsatellite sequence varies between individuals
  • Microsatellites are distributed across the whole genome - most are not within genes.
35
Q

How do you detect microsatellites in Qf-PCR?

A
  1. Isolate DNA from individual
  2. Design primers specific to flanking sequences
  3. PCR amplification
  4. Gel electrophoresis
  5. PCR amplification of microsatellite region
  6. Genotype size of fragments on gel-based system
    a. Homozygotes = single product of specific size
    b. Heterozygotes = two different sized products
36
Q

What is PCR?

A

Exponential amplification of a DNA fragment of known sequence

37
Q

State the key components of the PCR reaction?

A
  • Components of the PC reaction:
  • Template - DNA to amplify
  • Primers - Short pieces of SSDNA (15-30bp)
  • Polymerase - thermostable enzyme (Taq)
  • Nucleotides - single base mixture (dNTPs)
  • Buffer - To maintain pH
  • MCI2 - Essential for polymerase activity
38
Q

State the stages of the PCR cycle?

A
  1. Denaturation - heat and separate DNA strands at 94 degrees
  2. Annealing - Primers anneal with template (50-65 degrees approx. 60)
  3. Extension - DNA polymerase extends strand from primer (72 degrees)
    - PC exponential amplification occurs until components of reaction run out causing the reaction to slow down and stop
39
Q

Describe the graph and results of Qf-PCR on chromosome 21?

A
  • Perform PC using primers for microsatellite known to be on chromosome 21 (if testing for Down’s)
  • Should be two copies of microsatellite (one from mother, one from father, like any other autosomal locus, gene, whatever)
  • If homozygous, there will be a single peak of high signal
  • If heterozygous, there will be two peaks of similar, lower signal
40
Q

What is NIPT and NGS?

A
  • NIPT = Non-invasive pre-natal testing
  • NGS = Next generation sequencing
41
Q

What are the purposes of this technique?

A
  • Main purpose: ‘High chance’ indicator for invasive test of aneuplodies
  • Uses cell free fetal (extracted) DNA from maternal blood sample
  • Utilises next generation sequencing
  • Screening not diagnostic

Molecular Basis Of Disease (30 decks)

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